Development of an innate immune cell tolerance model

Abstract

Sepsis has been defined as the ‘life-threatening organ dysfunction caused by a dysregulated host response to infection’ carrying a poor prognosis and high mortality rates, both during its progression, as well as after hospital discharge, rendering it quite the adversary to modern medicine. One contributory aspect to this high mortality rates is sepsis-induced immune suppression, where cells of the innate immune system enter a state of tolerance and functional reprogramming, dampening any subsequent immune reaction to secondary infections. The work done in this dissertation aimed to develop an in vitro model of immune tolerance that serves as a standard on which future experimentation may be carried out. This was done by isolating and purifying human primary monocytes from young blood donors, characterising them using CD14, HLA-DR and FACS flowcytometry. Subsequently, a re-stimulation protocol was applied using bacterial LPS in order to induce endotoxin tolerance. The success in generating the tolerant state was assessed following the generation of significant differences in the mean cytokine secretion levels of TNF-α, IL-1β and IL-10 using ELISA and statistical computation, as well as through the confirmation of a retention in overall cell viability throughout using 7-AAD. It was found that a tolerant state was induced within the monocytes and macrophages, with TNF-α producing the most significant difference in mean secretion concentrations between the first and second stimulation, generating a pvalue of 7.824x10-4 . The differences in IL-1β and IL-10 secretion levels were 0.000542794 and 0.018214324, respectively. Following 7-AAD analysis, the overall cell viability was retained, indicating the successful acquisition of a tolerant state within the CD14+ , HLADR+ cell population.